Compositions and methods for ex vivo hepatic nucleic acid delivery

Abstract

The invention provides compositions and methods for delivery of nucleic acids to the liver ex vivo. The method includes slow, low pressure infusion of the nucleic acid into the liver to efficiently transduce cells with minimal cell damage.

1 . A method of ex vivo hepatic nucleic acid delivery comprising:
 obtaining a liver; and   injecting the nucleic acid in a carrier into vasculature of the liver at a low pressure 2-12 mmHg, whereby the nucleic acid is delivered to hepatic tissue.   
     
     
         2 . The method of  claim 1 , wherein the volume of the nucleic acid in the carrier is about 0.33 ml to about 20 ml per kg of liver to receive the nucleic acid in carrier. 
     
     
         3 . The method of  claim 1 , wherein the nucleic acid in the carrier is injected at a rate of 5-15 ml/minute. 
     
     
         4 . The method of  claim 1 , wherein the method further comprises perfusing the liver with cold pharmaceutically acceptable prior to the injection of the nucleic acid. 
     
     
         5 . The method of  claim 1 , wherein the nucleic acid in the carrier is injected into the portal vein, hepatic artery, or inferior vena cava. 
     
     
         6 . The method of  claim 1 , wherein the nucleic acid in the carrier is retained in the liver for at least 15 minutes. 
     
     
         7 . The method of  claim 1 , wherein the nucleic acid comprises an siRNA. 
     
     
         8 . The method of  claim 1 , wherein the nucleic acid comprises an antisense nucleic acid. 
     
     
         9 . The method of  claim 1 , wherein the nucleic acid comprises an shRNA. 
     
     
         10 . The method of  claim 1 , wherein the nucleic acid is targeted to a hepatitis virus. 
     
     
         11 . The method of  claim 10 , wherein the hepatitis virus comprises a hepatitis C virus. 
     
     
         12 . The method of  claim 11 , wherein at least a portion of the nucleic acid specifically hybridizes to at least 15 contiguous nucleic acids of a sequence selected from the group consisting of: 
       
         
           
                 
                 
               
                   5′-CUAGCCAUGGCGUUAGUAUUU-3′, 
                   (SEQ ID NO: 1) 
                 
                     
                 
                   5′-UCAACUGACUCGACCACUA-3, 
                   (SEQ ID NO: 2) 
                 
                     
                 
                   5′-GGAAGGUGCUUGUGGAUAUUU-3′, 
                   (SEQ ID NO: 3) 
                 
                     
                 
                   5′-GGGCCUAGGACCUGUAGUA-3′, 
                   (SEQ ID NO: 4) 
                 
                     
                 
                   5′-GCGAAGGCGUCCACAGUUA-3′, 
                   (SEQ ID NO: 5) 
                 
                   and 
                     
                 
                     
                 
                   5′-AGUCACGGCUAGCUGUGAAUU-3′. 
                   (SEQ ID NO: 6) 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         13 . The method of  claim 9 , wherein the antisense nucleic acid or the shRNA is encoded by an expression construct. 
     
     
         14 . The method of  claim 13 , wherein the expression construct is present in a vector selected from the group consisting of a plasmid vector, an adenoviral (Ad) vector, an adeno-associated viral vector (AAV), a lentiviral vector, and a herpes simplex viral (HSV) vector. 
     
     
         15 . The method of  claim 14 , wherein the AAV viral vector is selected from the group consisting of an AAV2 viral vector, an AAV8 vector, an AAV9 vector, a hybrid AAV2/4 viral vector, and a hybrid AAV2/5 viral vectors. 
     
     
         16 . The method of  claim 14 , wherein nucleic acid in the AAV viral vector is self-complementary. 
     
     
         17 . The method of  claim 14 , wherein the viral vector is replication competent. 
     
     
         18 . The method of  claim 14 , wherein the viral vector is replication incompetent. 
     
     
         19 . The method of  claim 13 , wherein the expression construct comprises a promoter sequence selected from the group consisting of a cytomegalovirus (CMV) promoter, a β-globin promoter, chicken β-actin (CBA) promoter, and small chicken β-actin (smCBA) promoter, and an EF-1α promoter. 
     
     
         20 . The method of  claim 14 , wherein the number of viral particles delivered comprises 10 10 -10 14  particles per 1.5 kg liver. 
     
     
         21 - 23 . (canceled) 
     
     
         24 . The method of  claim 1 , further comprising transplanting the liver into a subject. 
     
     
         25 . The method of  claim 24  further comprising monitoring the subject for HCV infection. 
     
     
         26 - 29 . (canceled)